JPH1192389A - Immunostimulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunostimulator capable of increasing the production of immunoglobulin A by an oral administration method, capable of stimulating the immunological activities of intestinal tracts and useful for foods used for preventing infectious diseases and the like by including the cells of a bacterium belonging to the genus Enterococcus as an active ingredient. SOLUTION: This immunostimulator contains the cells of a bacterium belonging to the genus Enterococcus, its died cells, or its bacteriolytic products or crushed products by a chemical, enzymatic or physical treatment as an active ingredient. The immunostimulator is obtained e.g. by inoculating Enterococcus faecalis NF-1011 strain in a ROGOSA liquid culture medium, culturing the bacterium at a temperature of 37 deg.C for 10-16 hr, centrifuging the culture liquid, washing the collected cells with distilled water, dispersing the cells in distilled water, heating the suspension at 110 deg.C for 10 min, lyophilizing the killed cell suspension, and subsequently using the obtained dried died cells as an active ingredient. The immunostimulator is preferably administered at a daily active ingredient dose of 1-100 mg/kg body for an adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention activates B cells that produce immunoglobulin A (IgA) in Peyer's patches, T cells that activate B cells, or antigen-presenting cells to prevent infectious diseases. The present invention relates to an orally administrable preparation that enhances the production of IgA having an action of suppressing the occurrence of harmful inflammation.

[0002]

2. Description of the Related Art It is well known that the boundary between a living body and the outside is the skin and mucous membrane, but the mucosal area is much larger. Among them, the intestinal mucosal surface has the largest area and is a place where many viruses, bacteria, parasites, pathogenic antigens and food antigens enter. Therefore, the intestinal mucosa has an intestinal immune system for protecting the body from these foreign antigens.

[0003] IgA is known to have an action of preventing the invasion of pathogenic substances and foreign proteins. This IgA
Plays an important role in neutralizing bacteria and viruses, suppressing bacterial adhesion to tissues, and suppressing allergy caused by food antigens.

[0004] The production of IgA is largely related to B cells, T cells and antigen presenting cells, which are leukocytes in Peyer's patches. T cells and antigen presenting cells have the effect of activating B cells. It is already known that an increase in the number of activated B cells increases the production of IgA.

[0005]

It is known that an infant, an elderly person, or a person with reduced physical strength has weak immunity and has a low resistance to foreign substances such as pathogenic bacteria. Therefore, there is a demand for a food having an action of maintaining a high level of IgA production, which plays an important role in neutralizing bacteria and viruses, suppressing the adhesion of bacteria to tissues, and suppressing allergy caused by food antigens.

[0006]

Means for Solving the Problems The present inventors have focused on a fungus, a bacterium belonging to the genus Enterococcus, which has an action of enhancing the defense against infection against fungi, bacteria and viruses. An experiment was performed using the cells, and as shown in Example 2, an increase in the amount of IgA produced from the cells constituting the Peyer's patch was observed. Furthermore, the same effect was observed in the case of oral administration, thereby completing the present invention.

[0007] As shown in Example 3, the enhancement of the blastogenesis reaction, which is an indicator of the activity of lymphocyte cells contained in Peyer's patches, was also observed in a concentration-dependent manner. It is also shown that the reactivity to foreign substances has increased. It is also considered that an adjuvant effect can be obtained.

The lactic acid bacterium used in the present invention is a strain that is edible or inhabits the intestinal tract of a healthy person, and therefore has no risk of side effects. For actual administration, live cells, dead cells, or cells obtained by grinding, extracting, or lysing the cells can be used. In order to make these into tablets, tablets, carriers, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like such as starch, lactose, soybean protein, etc. And granules.

[0009] The amount used depends on symptoms, age, etc.
Usually, 1 to 100 mg / kg body weight can be administered to an adult once or several times a day as an active ingredient.

[0010]

【Example】

Embodiment 1 FIG. Preparation of bacterial cell preparation Enterococcus faecali
s) NF-1011 (Microtechnological Laboratory No. 12564) was inoculated into Rogosa liquid medium having the following composition (the number of bacteria: 10 ^6).
Cells / ml) at 37 ° C. for 10 to 16 hours to obtain a culture solution with about 10 ⁹ viable cells / ml. The obtained culture solution was
The cells were collected by centrifugation at 2,000 xg for 20 minutes, and
The cells were washed twice to obtain cells. The cells are suspended in distilled water,
The suspension was heated at 10 ° C. for 10 minutes to prepare a dead cell suspension, and then dried by freeze-drying to obtain dried dead cells (hereinafter referred to as a cell sample).

The composition of Rogosa liquid medium is shown. Trypticase 10 g Yeast extract 5 g Tryptose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water 1000 ml (adjusted to pH 7.0, 121 ° C. 15 minutes heat-sterilized in) (1) _{saline: MgSO 4 · 7H 2 O 11.5g} FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled 100 ml of water

Embodiment 2 FIG. I from mouse Peyer's patch cells
gA production promoting action A Peyer's patch was removed from a male BALB / c mouse, 8-12 weeks old (Japan SLC), and an enzyme (Displace II:
Boehringer Mannheim).
5 × 10 ⁶ cells were prepared in RPMI medium supplemented with 10% FCS, and a monolayer was formed on a 96-well flat bottom plate. As an IgA inducer, 0.1, 10, 100, or 1000 μg / ml of the bacterial cell preparation prepared in Example 1 was added,
Mixed culture was performed for 7 days. After completion of the mixed culture, the supernatant was collected and
The gA amount was measured.

The obtained IgA was measured by an ELISA method. The measuring method is described in Robert. L. The method of Coffman et al. (Journal of Experimental Medicine vol. 170 pp:
1039-1044 (1989)).

FIG. 1 shows the results of the specific activity when the value of the control group was set to 1 when the cells were cultured without any addition to Peyer's patch cells. The bacterial sample increases IgA production in a concentration-dependent manner,
The specific activity when adding 100 or 1000 μg / ml is 1.
5, 1.8, which is significant from Peyer's patch cells (p <0.
05), an increase in IgA production was observed.

Embodiment 3 FIG. Lymphocyte blastogenesis of mouse Peyer's patch cells The Peyer's patch monolayer used in Example 2 was used.
After mixed culture with the cell sample, the MTT reagent was added to the Peyer's monolayer after collecting the supernatant. Two hours later, Peyer's patch cells took in the MTT reagent to extract the produced dye, and measured the absorbance at a wavelength of 588 nm to measure the blastogenesis reaction.

FIG. 2 shows the results of the specific activity when the value of the control group was set to 1 when the cells were cultured without any addition to Peyer's patch cells. The bacterial sample enhances the blast transformation in a concentration-dependent manner,
When 100 and 1000 μg / ml were added, the values of the specific activities were significantly significant (p <0.
05).

Embodiment 4 FIG. Oral administration enhances IgA production Three-week-old male BALB / c mice (Japan SLC) were divided into seven mice, one of which was CE-2 (Clear Japan) containing 5% of the bacterial cells prepared in Example 1. (Bacterial specimens).
The other received only CE-2 (control group). This feed was given until the end of the experiment.

Over time, the mice were placed in a metabolic gauge to collect feces, and the obtained feces were freeze-dried. The lyophilized stool was suspended in a buffer to a concentration of 10 mg / ml. The suspension was centrifuged at 700 × g for 5 minutes at 4 ° C. to recover the supernatant, which was used as an IgA sample. This sample was diluted to an optimum concentration and measured by an ELISA method. E
The LISA method was performed in the same manner as in Example 2.

FIG. 3 shows the change over time in the amount of IgA in feces. The amount of IgA in feces at the start of administration was 1 g of feces in both groups.
Approximately 150 ng. The IgA level increased over time until 5 weeks after the start of administration, but no difference was observed between the two groups. However, after 10 weeks from the start of administration, IgA in feces of the bacterial sample group increased compared to the control group, and the IgA amounts of the bacterial sample group and the control group increased to 1200 at week 10, respectively.
± 430 ng and 630 ± 370 ng, 1400 ± 800 ng and 540 ± 130 ng at 13 weeks, and 120 at 15 weeks (mouse age: 18 weeks) at the end of the experiment
0 ± 310 ng and 610 ± 220 ng (p <
0.05).

Embodiment 5 FIG. Enhancement of IgA production by treated lactic acid bacteria After recovering Enterococcus faecalis NF-1011 cells cultured according to the method of Example 1, the cells were suspended in distilled water, and mutanolysin was added at 30 μg / ml (final concentration).
After treatment at 37 ° C. for 4 hours, the mixture was heated at 110 ° C. for 10 minutes.
This cell suspension was dried by a freeze-drying method to prepare a processed product of lactic acid bacteria.

Based on the method of Example 4, 10 male BALB / c mice (Japan SLC) were bred in each group. Three weeks after the start of the administration, the amount of IgA in 1 g of feces of the group to which the lactic acid bacterium-treated product was administered was 1400 ± 270 ng, which was significantly (p <0.0) compared to 970 ± 340 ng of the control group.
A high value was obtained in 5).

[0022]

Since the amount of IgA produced by oral administration has been increased, it is expected that ingestion of this food will help prevent infectious diseases and the like. Moreover, since the amount of increase in IgA in feces is larger than that in the control group from childhood, it can be said that there is also an effect of promoting intestinal immunity.

[Brief description of the drawings]

FIG. 1 is a graph showing IgA production of mouse Peyer's patch cells in terms of specific activity.

FIG. 2 is a graph showing lymphocyte blastogenesis of mouse Peyer's patch cells in terms of specific activity.

FIG. 3: Fecal Ig when a bacterial sample was administered to mice
It is the graph which showed the amount of A.

Claims (3)

[Claims] 1. An immunostimulant comprising, as an active ingredient, a cell, a dead cell, or a lysate or crushed product obtained by chemical / enzymatic or physical treatment belonging to the genus Enterococcus. 2. The immunostimulant according to claim 1, which has an effect of enhancing immunoglobulin A producing ability by oral administration. 3. The immunostimulant according to claim 1, which has a blastogenic effect on leukocytes in Peyer's patches.